High-throughput LC/MS characterization of mRNA therapeutics using a fast DDA method on the Orbitrap Astral MS

Importance of the Topic

The rapid development of mRNA therapeutics, including vaccines and treatments for genetic disorders, demands robust analytical workflows. High-throughput liquid chromatography–mass spectrometry (LC-MS) offers detailed sequence confirmation, impurity detection, and coverage mapping essential for quality control and regulatory compliance.

Objectives and Study Overview

This study aimed to establish an end-to-end workflow for mRNA characterization. Key goals included optimizing partial enzymatic digestion, developing a fast data-dependent acquisition (DDA) LC-MS method, and implementing automated data analysis to achieve comprehensive sequence coverage of mRNA fragments.

Methodology and Instrumentation

The workflow combined the following steps

• Sample digestion: Partial cleavage of mRNA using RNase T1 immobilized on magnetic beads for defined time intervals (5–20 minutes)
• Chromatography: Separation of digestion fragments on a DNAPac RP column using a 15- to 30-minute gradient with TEA/HFIP buffers
• Mass spectrometry: Data-dependent MS/MS acquisition in negative ion mode on an Orbitrap Astral mass spectrometer with stepped normalized collision energies (three-level CE)
• Data processing: Automated fragment identification, annotation, and sequence coverage mapping using BioPharma Finder 5.2 software

Main Results and Discussion

The method achieved clear separation of oligonucleotides by size and charge, with higher retention for larger fragments. Stepped collision energy enhanced fragment diversity, improving confidence in sequence assignments. For a 3 500-base mRNA sample, the workflow delivered

• 88 percent coverage based on unique fragment identifications
• 92 percent total coverage when including non-unique fragments

Comparable experiments on a previous generation Orbitrap Exploris instrument yielded three times fewer MS2 scans, demonstrating the Astral’s superior acquisition speed (up to 45 Hz).

Benefits and Practical Applications

This approach provides a reproducible and high-throughput solution for:

• Sequence verification of mRNA therapeutics
• Detection of low-abundance impurities
• Quality control during manufacturing and method development

Future Trends and Opportunities

Advancements in MS instrumentation and data processing are expected to drive:

• Higher acquisition speeds and improved sensitivity for larger RNA molecules
• Deeper integration of AI-driven spectral interpretation
• Expanded workflows for intact mRNA analysis and modification mapping

Conclusion

The presented workflow demonstrates a fast, reliable, and automated LC-MS platform for comprehensive characterization of mRNA therapeutics. By combining optimized digestion, rapid chromatography, high-speed DDA acquisition, and automated analysis, laboratories can achieve near-complete sequence coverage and detect critical impurities with high confidence.

References

1. Diedrich JK, Pinto AFM, Yates JR. Energy Dependence of HCD on Peptide Fragmentation: Stepped Collisional Energy Finds the Sweet Spot. J Am Soc Mass Spectrom 2013.
2. Wei B et al. Development of an Ion Pairing Reversed-Phase LC-MS Method for Characterization of CRISPR Guide RNA. J Chromatogr A 2022.
3. Vanhinsbergh CJ et al. Characterization and Sequence Mapping of Large RNA and mRNA Therapeutics Using Mass Spectrometry. Anal Chem 2022.
4. Criscuolo A et al. Characterization of mRNA Therapeutics Using a Novel LC-MS Based Workflow. ASMS 2021.